期刊
BIOCHEMISTRY
卷 60, 期 25, 页码 1971-1982出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.1c00270
关键词
-
资金
- National Institutes of Health (NIH) [R35 GM122532, R01 AI068462, R35 GM134864, R01 GM101237, R01 HL111527, T32 GM067553, T32 GM008570, T32 GM08570]
- Medical Research Council [MC_U105178804]
- Passan Foundation
- National Science Foundation [DGE-1144081]
The SHAPE-JuMP technique utilizes chemical reactions and engineered reverse transcriptase to rapidly and accurately reveal the three-dimensional interactions of large RNA molecules in solution, particularly effective for measuring interactions in multihelix junctions and loop-to-helix packing, enabling easier global folding modeling and structural model ranking.
Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. A bifunctional small molecule is used to chemically link proximal nucleotides in an RNA structure. The RNA cross-link site is then encoded into complementary DNA (cDNA) in a single, direct step using an engineered reverse transcriptase that jumps across cross-linked nucleotides. The resulting cDNAs contain a deletion relative to the native RNA sequence, which can be detected by sequencing, that indicates the sites of cross-linked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity concisely across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions in multihelix junctions and loop-to-helix packing, enables modeling of the global fold for RNAs up to several hundred nucleotides in length, facilitates ranking of structural models by consistency with through-space restraints, and is poised to enable solution-phase structural interrogation and modeling of complex RNAs.
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